唾液酸酶及固醇硫酸酶活性探針在生物醫學上的應用

Biomedical Application of Activity Probe for Neuraminidase and Steroid Sulfatase

呂君萍(Chun-Ping Lu)

指導教授 ： 吳世雄

國立臺灣大學/生命科學院/生化科學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.02487

摘要

以酵素的活性為基礎的而設計的小分子活性探針可以用來標示、濃縮、分離及區分蛋白質。它會選擇性的與酵素反應並共價鍵結於酵素上。活性探針的組成包含四個主要部分：辨識端、捕捉機制、連結鏈與發報端。在本研究中，活性探針6、11及12被用來測試標定流感病毒唾液酸酶及固醇硫酸酶。以活性探針12為基礎而設計的化合物17、18及19則用以抑制固醇硫酸酶的活性。
[第一部分]
爲了偵測及抑制流行性感冒病毒唾液酸酶，探針6的結構包含四個部分：唾液酸辨識端、quionoe methide捕捉機制、連結鏈及用以偵測分離的生物素發報端。一但唾液酸酶水解唾液酸，所產生的高活性quinone methide便會共價修飾酵素。在本研究中，我們以西方墨點法觀察到生物素標示的Arthrobacter ureafaciens唾液酸酶。針對病毒捕捉的實驗，探針藉由卵白素與生物素的交互作用固定於微孔盤上，並利用酵素免疫分析法證實可以成功地捕捉到流行性感冒病毒。合併反轉錄-聚合酶鏈反應與專一性的引子對可成功的區分被捕捉的流行性感冒病毒種類。本研究這個新的方法提供了一個機會可以快速利用抗體篩選流行性感冒病毒及發展更敏感快速的診斷方法。
[第二部分]
固醇硫酸酶藉由調控estrone sulfate的脫硫反應促進乳癌細胞吸收estrogen。人類固醇硫酸酶被發現在乳癌腫瘤上有表現量及活性增加的趨勢也因此成為藥物發展的標的。在本研究中，兩種固醇硫酸酶活性探針(探針11及12)及三種抑制劑(化合物17、18及19被用來標示及抑制固醇硫酸酶。標示的研究說明了探針11會專一性的與固醇硫酸酶反應。墨點分析也說明了探針11所標示的訊號強度與酵素的活性有關。更進一步的研究發現，探針12比探針11具有更高的特異性與較少的擴散反應發生。在抑制的研究方面，三價的酪氨酸硫酯化衍生物，化合物19，顯示有較化合物17和18更好的抑制能力。活細胞的實驗也證明了化合物19對倉鼠卵巢細胞具有適當的通透性及不具細胞毒性的特性。對於發展乳癌的治療固醇硫酸酶探針12及化合物19將會是一個有用的化學方法用於固醇硫酸酶的鑑別、抑制劑的設計及蛋白質體內標的物功能性狀態的決定。

關鍵字

流行性感冒病毒 ； 活性探針 ； 唾液酸酶唾液酸 ； 生物素 ； 固醇硫酸酶抑制劑

並列摘要

Depending on their enzymatic activities, the small molecular activity probe can be used to tag, enrich or isolate distinct sets of proteins. It only selectively reacts with enzymes that have a catalytic activity and covalent linkage with targeted enzyme. Activity probe contains four major components: a recognition head, a trapping device, a linker and a reporter group. The designed probe 6 was employed by this study to test its labeling effects on influenza virus neuraminidase (NA), whereas for the application of steroid sulfatase (STS), probes 11 and 12 were adopted. Compounds 17, 18 and 19, whose structures were designed on the basis of that of probe 12, were used for inhibiting STS activity.
Part I
For the detection and inhibition of influenza virus NA, the structure of probe 6 was designed to contains four fragments, a sialic acid for recognition, a latent quinone methide for trapping device, a linker and a biotin as reporter for detection and separation. Once a sialic acid is released by the hydrolysis of NA, the enzyme can be covalently modified by the resulting highly reactive quinone methide. In our study, the biotin labeled Arthrobacter ureafaciens NA were observed by Western blotting. For virus capturing experiment, the probe was be used to attach the microplates through avidin-biotin interactions and the captured virus were successfully demonstrated by the ELISA assay. To identify the captured virus, RT-PCR method combined with specific primer sets was future implemented. Overall, the novel approach adopted in this study offers opportunities for the rapid screening of antibodies against influenza virus and development of sensitive, rapid diagnostic methods.
Part II
Steroid sulfatase (STS) facilitates the estrogen uptake of breast cancer cells by mediating the desulfation of estrone sulfate. Human steroid sulfatase has become a target for drug development due to its increased expression and activity in breast carcinoma. In this study, two activity probes (probes 11 and 12) and three inhibitors (compounds 17, 18, and 19) were used for STS labeling and inhibition. Regarding STS labeling, probe 11 was shown to specifically react with STS. The dot signal intensity of probe 11 was illustrated by dot blot analysis to be correlated with enzymatic activity. Compared with probe 11, probe 12 demonstrated that higher specificity and less cross-reactivities. For STS inactivation study, compound 19, trivalent tyrosine sulfate ester derivative, revealed better inhibition ability than compounds 17 and 18. For compound 19, moderate cell permeability and no cytotoxicity for CHO cells were demonstrated in live cell assay. As a result, for the development of breast cancer treatment, probe 12 and compound 19 may be two effective chemical devices in STS identification, STS inhibition, and functional state determination in complex proteome.

並列關鍵字

influenza virus ； activity probe ； quinone methide ； neuraminidase ； sialic acid ； biotin ； steroid sulfatase ； inhibitor

參考文獻

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